USE OF TREZASTILBENOSIDE IN MANUFACTURE OF PRODUCT FOR TREATING AND/OR PREVENTING NON-ALCOHOLIC FATTY LIVER DISEASE

Abstract

The invention belongs to the technical field of biopharmaceuticals, and discloses the application of Trezastilbenoside in the manufacture of products for treating and/or preventing non-alcoholic fatty liver disease. The research of the present invention shows that after giving Trezastilbenoside to non-alcoholic fatty liver disease (NAFLD) model mice for 4 consecutive weeks, the serum TC, TG, and LDL content were all significantly reduced, and the HDL content was significantly increased, indicating that the drug has a lipid regulatory effect. The activities of AST and ALT in the serum were significantly weakened, and the infiltration of inflammatory factors in the liver tissue was reduced, indicating that the drug has a hepatoprotective effect. It can not only reduce the fat content of the liver, but also improve the pathological form of fatty liver, indicating an effect of against NASH.

Claims

1. A method of treating and/or preventing non-alcoholic fatty liver disease, comprising administering Trezastilbenoside to a subject in need thereof.

2. The method of claim 1, comprising administering Trezastilbenoside to the subject to improve the increase in body weight and/or liver weight of the subject.

3. The method of claim 1, comprising administering Trezastilbenoside to the subject to improve the increase in total cholesterol and/or triglycerides in serum of the subject.

4. The method of claim 1, comprising administering Trezastilbenoside to the subject to improve the increase in low density lipoprotein in serum of the subject.

5. The method of claim 1, comprising administering Trezastilbenoside to the subject to improve the increase in aspartate aminotransferase and/or alanine aminotransferase in serum of the subject.

6. The method of claim 1, comprising administering Trezastilbenoside to the subject to improve the decrease in high density lipoprotein in serum of the subject.

7. The method of claim 1, wherein the product includes a food product, a health product and a medicament.

8. The method of claim 1, wherein the product for treating and/or preventing non-alcoholic fatty liver disease is a medicament, comprising Trezastilbenoside and a pharmaceutically acceptable adjuvant.

9. The method of claim 8, wherein the medicament for treating and/or preventing non-alcoholic fatty liver disease is provided as various acceptable dosage forms; preferably, the dosage forms include injections, tablets, capsules, powders, pills or oral solutions.

10. A product for treating and/or preventing non-alcoholic fatty liver disease, comprising Trezastilbenoside; preferably, the product includes a food product, a health product and a medicament; and preferably, the product is a medicament comprising Trezastilbenoside and a pharmaceutically acceptable adjuvant.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0031] The accompanying drawings are used as a part of the present disclosure to provide a further understanding of the present disclosure. The exemplary embodiments and descriptions of the present disclosure are used to explain the present disclosure, but do not constitute an improper limitation of the present disclosure. Apparently, the drawings in the following description are only some embodiments. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without inventive work. In the drawings:

[0032] FIG. 1 is an image showing the pathological changes in mouse liver under a microscope (HE, ×200) of the present disclosure,

[0033] wherein A is the normal control group, B is the model group, C is the Trezastilbenoside low dose group, and D is the Trezastilbenoside high dose group.

[0034] It should to be noted that these drawings and the word description are provided to explain the concept of the present disclosure by referring to particular examples for these skilled in the art, but are not intended to limt the scope of the concept of the present disclosure in any way.

DETAILED DESCRIPTION

[0035] In order to make the purposes, technical solutions and advantages of examples of the present disclosure clearer, hereafter the technical solutions in the examples will be described clearly and completely in conjunction with the drawings. The following examples are given to describe the present disclosure, and are not intended to limit the scope of the present disclosure.

Example 1: The Protective Effect of Trezastilbenoside on Non-Alcoholic Hepatocyte Injury

1. Experimental Materials

1.1 Testing Samples

[0036] Trezastilbenoside, with a molecular weight of 406, white crystalline or crystalline powder, 99.6% purity and a lot number of 20120402.

1.2 Experiment Animals

[0037] 44 healthy male C57BL/6 mice with a body weight of 19-23 g, purchased from Hunan SJA Laboratory Animal Co., Ltd with Laboratory Animal Production License No. SCXK(XIANG)2016-0002, were housed in groups inside PVC transparent plastic boxes of 6 mice or less in each box, at Research Institute of KPC Pharmaceuticals, Inc. The mice were given corresponding feed every day and received water ad libitum, with the cages and bedding changed when needed, at a temperature of 20-25° C. (daily temperature difference was ≤3° C.), a humidity of 40%-70%, under the illumination in a mode of 12 h: 12 h light/dark with an illumination intensity of 150-300 lx, a noise of 60 dB or less, and with a Laboratory Animal Use Permit NO. SYCK (DIAN) K2019-0001 issued by the Kunming Science and Technology Bureau.

1.3 Feed

[0038] A high-sugar and high-fat feed D09100310 containing 40% of fat, 20% of fructose and 2% of cholesterol, from Shuyishuer Bio-Technology Co., Ltd, was irradiated at 15 kGy;

[0039] An ordinary feed was from Jiangsu Xietong Medical Bioengineering Co., Ltd., with a permit No. Susizheng (2014) 01008.

1.4 Reagents and Instruments

[0040] Paraformaldehyde (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), disodium hydrogen phosphate (analytical grade, Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.), and sodium dihydrogen phosphate (analytical grade, Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.) were employed. Reagent kits such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), low density lipoprotein (LDL) and high density lipoprotein (HDL) were all original reagents produced by Nanjing Jiancheng Bioengineering Institute. Xylene, absolute ethanol, formaldehyde, acetone, hematoxylin, Sudan IV, eosin, hydrochloric acid, ethanol, neutral gum, glycerin, gelatin, etc. were reagents required by pathological testing. Picric acid, sodium chloride injection solution, disinfection alcohol, etc. were common reagents used in laboratories. 96-well flat-bottom cell culture plates, embedding cassettes, paraffin wax, glass slides, cover glasses, slicing knives, etc. were used.

[0041] A tissue homogenizer, a high-speed freezing centrifuge, an microplate reader, an electronic scale, and an analytical balance; a dewaterer, LEICA ASP300S; a freezing microtome, LEICA CM1950; an embedding machine, LEICA EG1150H; a freezing table, LEICA EG1130; a rotary section machine, LEICA RM2235; a dryer, LEICA HI1220, etc. were employed.

2. Test Methods

2.1 Preparation of Animal Models

[0042] After 1 week of adaptive feeding, 44 C57 mice were divided into a control group (12 mice) and a model group (32 mice) according to the random number method. The normal control group was given ordinary diet, and the model group was given high-sugar and high-fat diet. After feeding for 8 weeks, randomly select 2 animals in each of the control group and the model group. After pathological examination confirmed that the modeling was successful, the model animals were divided into groups.

2.2 Grouping and Administration

[0043] The model animals were divided according to random number method into 3 groups of 10 mice each, namely model control group (equal volume of sodium chloride injection solution), Trezastilbenoside high dose group (20 mg/kg) and Trezastilbenoside low dose group (10 mg/kg). The mice in each administration group were administered intragastrically with the corresponding drug, once a day continuously for 4 weeks, while being fed with the high-sugar and high-fat feed continuously. The mice in the normal control group (equal volume of sodium chloride injection solution) were feed with the ordinary feed.

2.3 Observation Indicators

2.3.1 General Observation

[0044] After the last administration, the mice were fasted but had access to water for 12 h, and then sacrificed after blood was taken. The body mass and liver mass of each mouse were weighed to calculate the liver index by the following formula.

liver index=(liver wet mass/body mass)×100% (wet liver mass/body mass).

2.3.2 Measurement of the Levels of Serum TC, TG, HDL, LDL, AST and ALT

[0045] About 1 mL of blood, taken via mouse eyeballs, was placed into a 1.5 mL centrifuge tube, and then centrifuged at 3000 r/min for 10 min to obtain the supernatant. The levels of TC, TG, HDL, LDL, AST and ALT in the serum were measured with a microplate reader strictly under the operating instructions of the assay kit.

2.3.3 Measurement of TC and TG Content in Liver Tissue

[0046] About 100 mg of liver tissue weighed, was put into 900 μL absolute ethanol to make a 10% tissue homogenate under an ice bath. Then the supernatant was separated out through a centrifugation separation at 3000 r/min for 15 min, and collected into a 1.5 mL centrifuge tube for the measurement of TC and TG content in the liver tissue.

2.4.4 HE Staining of Liver

[0047] The fresh liver tissue was immersed and fixed in 4% formaldehyde solution overnight for 24 h, dehydrated through 75%-95%-100% ethanol gradient, embedded in paraffin, sectioned into 4 μm, and conventionally stained with hematoxylin-eosin, to observe the pathological changes of the liver tissue under an optical microscope (HE, 200×).

3. Statistical Methods

[0048] Experiment data were statistically analyzed using SPSS 20.0 software. The measurement data were expressed as X±s. T test was used for comparison between groups. A P-value less than 0.05 means that the difference is statistically significant.

4. Experiment Results

4.1 Effects on the Body Weight, Wet Liver Weight and Liver Index as Well as the General Conditions of Mice

[0049] During the experiment, the mice in each group were in good condition and had a strong appetite. Among them, the mice in the normal group showed a more docile temperament, with smooth fur and normal activities; the mice in the NAFLD model group gained rapid weight, and showed more irritable temperament and did not like to move. After 12 weeks of high-sugar and high-fat feeding, compared with the normal control group (normal diet), the weight gain, liver wet weight and liver index of the model group (high-sugar and high-fat diet) were significantly higher than those of the normal control group (P<0.01); Compared with the model group, the Trezastilbenoside administration group has a tendency to reduce the obesity induced by high sugar and high fat in mice, and significantly reduced the wet weight and liver index of the model animals (P<0.05). The results are shown in the table 1.

TABLE-US-00001 TABLE 1 Comparisons of the body weight, liver weight and liver index of mice (  , g) Groups N Weight (g) Wet liver weight (g) Liver index (%) Normal group 10 25.7 ± 0.63 0.99 ± 0.22 0.38 ± 0.02 Model group 10 36.2 ± 0.55.sup.## 2.14 ± 0.21.sup.## 0.59 ± 0.14.sup.## Trezastilbenoside low dose group 10 35.0 ± 0.62 1.84 ± 3.0* 0.53 ± 0.0.4* Trezastilbenoside high dose group 10 33.8 ± 0.74 1.72 ± 2.7* 0.51 ± 0.05* Note: .sup.##P-value less than 0.01 as compared to the normal control group; *P-value less than 0.05 as compared to the model group

4.2 Comparisons of the Levels of TC, TG, HDL, LDL, AST and ALT in Serum

[0050] Compared with the normal control group, the serum levels of TC, TG, LDL and the activities of AST and ALT in the model group were significantly increased, and the HDL content was significantly reduced (P<0.05/0.01); compared with the model group, in the high-dose Trezastilbenoside group, the contents of TC, TG, LDL in the serum of mice were significantly reduced, the activities of AST and ALT were significantly weakened, and the content of HDL was significantly increased (P<0.01), indicating that Trezastilbenoside has a good protective effect on non-alcoholic fatty liver disease induced by high sugar and high fat. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Comparisons of the changes of TC, TG, HDL, LDL, AST and ALT in the mouse serum (x ± s, n = 10) TC TG HDL LDL AST ALT Groups (mmol/L) (mmol/L) (mmol/L) (mmol/L) (U/L) (U/L) Normal group 2.63 ± 0.27 0.77 ± 0.08 1.53 ± 0.14 0.49 ± 0.21 143.56 ± 61.80  97.86 ± 8.51 Model group 6.89 ± 0.67.sup.# 2.80 ± 0.21.sup.# 0.09 ± 0.50.sup.# 0.67 ± 0.30.sup.# 232.22 ± 46.90.sup.# 165.24 ± 10.52.sup.## Trezastilbenoside 5.53 ± 0.52* 2.67 ± 0.19 0.27 ± 0.67 0.47 ± 0.13* 147.73 ± 52.98* 122.45 ± 27.37* low dose group Trezastilbenoside 3.43 ± 0.17** 0.99 ± 0.11** 0.45 ± 0.16** 0.41 ± 0.30* 118.92 ± 10.75** 78.30 ± 11.53** high dose group Note: .sup.#P-value less than 0.05, .sup.##P-value less than 0.01, as compared to the normal control group; *P-value less than 0.05, **P-value less than 0.01, as compared to the model group.

4.3 Effects on the TC and TG Content in the Mouse Liver Tissue

[0051] Changes in liver fat content can better reflect the degree of fatty liver, and should be taken as one of the important indicators for judging the efficacy of anti-fatty liver drugs. After the mice were given the high-sugar and high-fat feed for 12 weeks, their liver TG and TC levels were all significantly increased. Trezastilbenoside can significantly reduce the liver TG and TC levels of fatty liver mice, which are statistically different from the model control group (P<0.05/0.01), the results are shown in Table 3.

TABLE-US-00003 TABLE 3 Effects on the lipid content in the mouse liver (  , mg/g) Groups TC (mg/g) TG (mg/g) Normal group 3.08 ± 0.54 1.34 ± 0.18 Model group 7.22 ± 0.85.sup.## 3.25 ± 0.81.sup.## Trezastilbenoside low dose group 6.53 ± 0.59* 2.12 ± 0.58 Trezastilbenoside high dose group 4.23 ± 0.87** 1.96 ± 0.99** Note: .sup.##P-value less than 0.01, as compared to the normal control group; *P-value less than 0.05, **P-value less than 0.01, as compared to the model group.

4.4 Gross Observation of Mouse Liver and Pathological Observation of Liver Tissue

4.4.1 Gross Observation of Mouse Liver

[0052] The liver of the mice in the normal control group was dark red, bright in color, soft and fragile. The liver was slightly wedge-shaped, the right end was round and thick, and the left end was narrow and thin without greasy feeling; the mouse liver in the model group was enlarged and yellow in color; the texture was hard, with granular and greasy feeling when pinched; the liver color of the mice in the low and high dose groups of Trezastilbenoside was between the normal control group and the model group, and the texture and shape tended to be normal.

4.4.2 HE Observation of Mouse Liver

[0053] HE staining, under the microscope, it was seen that the liver tissue structure of the normal group was complete and clear, and the liver lobule structure was normal. The liver cells were arranged in hepatic cords and distributed radially around the central vein. The center of the cells had a large round nucleus, and the cytoplasm was uniform and no fat droplet. In the model group, liver steatosis was obvious. The liver cells were enlarged and contained large fat droplets. Some cells were found cell nuclei squeezed into the cell membrane, showing a large vesicular steatosis, and inflammatory cell infiltration was found. Punctate necrosis and focal necrosis were found in some cells, showing progressed to the stage of non-alcoholic steatohepatitis. Compared with the model group, the fat droplets in the Trezastilbenoside group were smaller and fewer, the pathological morphology of liver tissue was significantly improved, the fatty degeneration of vacuolated liver cells were significantly reduced, the infiltration of inflammatory cells was not obvious, and the cell arrangement was relatively neat and complete. These showed that Trezastilbenoside reduced the infiltration of hepatic inflammatory cells caused by high sugar and high fat, as shown in FIG. 1.

[0054] FIG. 1 is the pathological changes of mouse liver under the microscope of the present invention (HE, ×200); among them, A is the normal control group, B is the model group, C is the Trezastilbenoside low dose group, and D is the Trezastilbenoside high dose group.

[0055] It can be seen from the experimental results in FIG. 1 that, after 4 consecutive weeks of administration of Trezastilbenoside to model mice, the serum levels of TC, TG, and LDL were significantly reduced, and the HDL content was significantly increased, indicating that the drug has a lipid-regulating effect. The activities of AST and ALT in the serum were significantly weakened, indicating that the drug has the effect of improving liver function; the pathological morphology of the liver showed improvement in liver tissue steatosis and reduce of the infiltration of inflammatory factors, indicating that the drug has the effect of protecting the liver. It has good clinical value in the prevention and treatment of non-alcoholic fatty liver (NAFLD).

Example 2

[0056] The tablet in this example was comprised of the following components: 10 g of Trezastilbenoside, 20 g of microcrystalline cellulose, 20 g of pregelatinized starch, 20 g of cross-linked polyvinylpyrrolidone, and 1 g of micropowder silica gel.

[0057] The above raw materials were mixed to obtain the Trezastilbenoside tablet in accordance with a conventional preparation method.

Example 3

[0058] The capsule in this example was comprised of the following components: 10 g of Trezastilbenoside, 30 g of microcrystalline cellulose, 5 g of lactose, a proper amount of povidone K-30, and 1 g of magnesium stearate.

[0059] The above raw materials were mixed to obtain the Trezastilbenoside capsule in accordance with a conventional preparation method.

Example 4

[0060] The granule in this example was comprised of the following components: 10 g of Trezastilbenoside, 20 g of mannitol, 20 g of lactose, 1 g of sodium cyclamate, 0.5 g of solid edible essence, and 1 g of xanthan gum.

[0061] The above raw materials were mixed to obtain the Trezastilbenoside granule in accordance with a conventional preparation method.

Example 5

[0062] The dropping pill in this example was comprised of the following components: 5 g of Trezastilbenoside and 15 g of polyethylene glycol 6000.

[0063] The above raw materials were mixed to obtain the Trezastilbenoside dropping pill in accordance with a conventional preparation method.

[0064] The above are only the preferred examples of the present disclosure, and do not limit the present invention in any form. Although the present invention has been disclosed as the preferred examples, it is not intended to limit the present invention. Those skilled in the art familiar with this patent, can make slight changes or modification into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present disclosure. For any simple modifications, equivalent changes and modifications made to the above embodiments based on the technology of the present invention without departing from the technical solution of the present invention, they still fall within the scope of the present invention.